Pigmentation enhancer and method

ABSTRACT

A composition for increasing synthesis of melanin in a human melanocyte thereby enhancing pigmentation of the human skin. Use of this composition promotes tanning of the human skin and increases photoprotection from ultraviolet radiation. An organ culture system comprising viable human foreskin samples which may be used to test the effects of agents on human skin, including pigmentation enhancers on human skin.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of co-pending U.S. Ser. No.07/943,998, filed Sep. 11, 1992, titled PIGMENTATION ENHANCER ANDMETHOD, and is a continuation of U.S. Ser. No. 07/451,420, filed Dec.15, 1989, titled PIGMENTATION ENHANCER AND METHOD, now abandoned.

FIELD OF THE INVENTION

The present invention generally relates to compositions for topicalapplication on a subject's skin, and specifically, to pigmentationenhancers, and to methods for assaying the effects of compositions onskin.

SUMMARY OF THE INVENTION

The present invention comprises a composition of matter comprising aneffective amount of a phosphodiesterase-inhibitor. Thephosphodiesterase-inhibitor is capable of increasing levels of melaninin a human melanocyte. The composition also comprises an effectiveamount of a pharmaceutically acceptable topical carrier. The carrier iscapable of delivering the phosphodiesterase-inhibitor to the melanocyteunder in vivo conditions.

A composition of matter of the present invention may also comprise aneffective amount of a methylxanthine which is capable of increasinglevels of melanin in a human melanocyte. The composition also comprisesan effective amount of a pharmaceutically acceptable topical carriercapable of delivering the methylxanthine to the melanocyte under in vivoconditions.

The present invention also comprises a method for at least partiallyshielding the skin from ultraviolet radiation in a subject. Thesubject's skin is treated with a pigment enhancer comprising an agentcapable of increasing the amount of melanin in a human melanocyte, and apharmaceutically acceptable topical carrier as previously described.

Another method of the present invention is a method of promoting tanningof a subject's skin. The skin is treated with a pigment enhancer aspreviously described.

The present invention is also directed to a human organ culturecomprising a viable human foreskin tissue specimen having an epidermisand a dermis, a nutrient medium, and a means for supporting the foreskinin the medium. The foreskin is supported such that the dermis contactsthe medium, and the epidermis is in substantially non-contactingrelationship with the medium.

The human organ culture may be used to assay the effects of agents onskin. A baseline level of a biological factor in the foreskin isobserved. The foreskin is contacted with the agent, and a post-contactobservation made of the biological factor in the foreskin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a plan view of the organ culture system of the presentinvention.

FIG. 2 is a side cross-section view of the organ culture system shown inFIG. 1 taken along line 2--2 shown in FIG. 1.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Skin color in humans arises from a complex series of cellular processeswhich are carried out within a unique population of cells calledmelanocytes. Melanocytes are located in the lower part of the epidermis,and their function is to synthesize a brown pigment, melanin, whichprotects the body from the damaging effects of ultraviolet radiation.

When skin is exposed to ultraviolet radiation, such as that contained insunlight, melanocytes increase their synthesis of melanin. Melanin isdeposited in melanosomes, which are vesicles found within the cell. Themelanosomes are extruded from the cell and carried to the surface of theskin by keratinocytes, which internalize the melanin containingmelanosomes. The end result is that the visible layers of the skinexhibit a brown color typically known as a "tan".

The darkness of the color observed in the skin is proportionate to theamount of melanin synthesized by melanocytes and transferred to thekeratinocytes. By increasing melanogenesis, skin can be at leastpartially shielded from ultraviolet radiation, and tanning of the skincam be achieved without exposure of the skin to potentially damagingultraviolet radiation. The present invention is directed to compositionsand methods which increase melanogenesis in subjects. "Subjects" as usedherein mean mammals, and, more preferably, humans.

The present invention comprises a composition of matter comprising aneffective amount of an agent capable of increasing the amount of melaninin a melanocyte (hereafter "melanin-increasing agent") and, morepreferably, in a human melanocyte. The melanin-increasing agent mayfunction in any of the various ways that are believed to increase theamount of melanin: by increasing cAMP or derivatives of cAMP whichfunction as cAMP in the melanocyte; by inhibiting phospho-diesterase; orby increasing tyrosinase activity. The melanin-increasing agent shouldbe capable, alone or with the aid of a carrier as described hereafter,of crossing the melanocyte membrane, and more preferably, a humanmelanocyte membrane, in order to function as will be described.

Preferably, the melanin-increasing agent of the present inventioncomprises at least one phosphosdiesterase-inhibitor.Phosphodiesterase-inhibitors are believed to increase the amount of cAMPin biological systems. cAMP is believed to increase the activity oftyrosinase, which is the rate limiting enzyme which transforms thesubstrate tyrosine into melanin.

More preferably, the melanin-increasing agent of the present inventioncomprises at least one metnylxanthine, and preferably a methylxanthinecapable of functioning as a phosphodiesterase inhibitor. Mostpreferably, the agent comprises theophylline, isobutyl methylxanthine,or a derivative thereof such as aminophylline. Additionally,melanin-increasing agents of the present invention may be combined inorder to enhance pigmentation of the individual.

The melanin-increasing agent may also comprise any pharmaceuticallyacceptable salt, analog, derivative or isomer of any phosphodiesteraseinhibitor or any methylxanthine capable of functioning as describedherein.

The composition of matter of the present invention additionallycomprises an effective amount of a pharmaceutically acceptable topicalcarrier capable of delivering the agent to the melanocyte under in vivoconditions. The carrier may comprise any solution, suspension, emulsionor any other form which is capable of delivering the agent to themelanocyte under in vivo conditions. "Capable of delivery", as usedherein, means that the carrier should permit the agent to cross thestratum corneum and successive cell layers found epidermal to themelanocyte, and/or aids the agent in crossing the melanocyte membrane.Furthermore, the carrier should not substantially interact with theagent so that the agent may perform its function as described herein.

The identity and quantity of the carrier will depend on the identity ofthe melanin-increasing agent used in the composition of the presentinvention. However, in many instances, the carrier will represent fromabout 95 percent to about 99 percent of the composition. In a preferredembodiment, the carrier may comprise 20% water, 50% isopropanol and 30%glycerol, combined with theophylline to produce a composition containing0.2 percent by weight theophylline. In another embodiment, the carriermay comprise 50% propylene glycol, 20% water and 30% isopropanol toproduce a composition containing 0.2 percent by weight theophylline.

The melanin-increasing agent is present in the composition of thepresent invention in any effective amount. An "effective amount" of themelanin-increasing agent is an amount which increases melanogenesis inthe treated area of the subject. This amount may vary with, among otherthings, the identity of melanin-increasing agent and carrier, thesubject's skin color and condition, and the degree of tanning and/orphotoprotection sought. Preferably, the melanin-increasing agent ispresent in the composition from about 0.01 mM to about 100 mM. In apreferred embodiment, 18 mg of theophylline may be combined with 10 mlof a pharmaceutically acceptable topical carrier to cover about a 50square inch area of skin. In another preferred embodiment, 180 mg oftheophylline may be combined with 10 ml of a pharmaceutically acceptabletopical carrier to form a composition capable of covering about a 50square inch area of skin. In another embodiment, 2.2 mg of isobutylmethylxanthine may be combined with 10 ml of a pharmaceuticallyacceptable carrier to cover about a 50 square inch area of skin.

As previously discussed, the enzyme tyrosinase converts the substratetyrosine into melanin. Since the composition of the present invention isbelieved to increase tyrosinase activity, the present inventionpreferably further comprises an effective amount of tyrosine so that thetyrosinase has as much substrate as possible to synthesize melanin. An"effective amount" of tyrosine means any amount which can be utilized bytyrosinase over the amount of tyrosine already present in themelanocyte. A preferable amount of tyrosine is about 0.01 mM to about 1mM of the composition of the present invention. In a preferredembodiment, 1.8 mg of tyrosine may be combined with 18 mg oftheophylline and 10 ml of a pharmaceutically acceptable topical carrier.

The composition of matter of the present invention is preferably applieddirectly to the skin of the individual seeking tanning of the skinand/or photoprotection from ultraviolet radiation. The treated area canbe the entire skin surface of the subject or only those areas normallyexposed to ultraviolet radiation. Since the composition of the presentinvention does not dye the skin, but rather permits the body chemistryto produce the tan, the tanning will not be streaked in the areas ofapplication. Application of the composition may be repeated periodicallyfor greater protection and/or tanning effect.

The composition of matter is preferably in a lotion or solution formwhich may be manually rubbed on the skin. Other means of application areacceptable such as aerosol sprays or the use of an applicator bottle.

ORGAN CULTURE SYSTEM

The adequate evaluation of the effects of an agent applied to human skinhas been difficult, if not impossible, due to the nature of the systemspreviously used. For example, to study the pigmentation process, mousemelanoma cells, human melanoma cells, and human melanocytes in culturetreated with tumor promotor agents have been used. The results ofexperiments using these abnormal cells were often inconsistent and didnot always correlate to the results obtained in the normal human skin.

Rather than use abnormal cells placed outside their normal environment,the present invention comprises cells in situ in an organ and maintainsthe viability of the organ in order to emulate the human skin in vivo.It is the development of a human organ culture system of the presentinvention which has permitted the adequate evaluation and development ofthe compositions and methods of treatment of the present invention.

The human organ culture of the present invention employs a viableforeskin. "Viable" means there has been no substantial morphologicalchange in the foreskin after surgical removal. Viability may bedetermined by changes in tissue ultrastructure determined throughhistochemical staining and/or dopa reaction staining, techniques whichpermit monitoring of any changes in the tissue ultrastructure.

The foreskins may be obtained by circumcising male neonates by standardsurgical procedures. After surgical removal, the foreskin is preferablyprepared for the organ culture by injection intradermally with themedium described hereafter. This swells the mucous membrane and allowsfor the removal of the membrane thereby allowing adequate nutrient flowto the foreskin through the dermis.

The foreskin comprises an epidermis which is normally exposed to theenvironment and a dermis opposing the epidermis. After surgical removaland preparation for the organ culture, the dermis, which is normallysupplied nutrients by the body, is exposed to the environment. In orderto maintain the viability of the foreskin, a nutrient medium suppliesnutrients to the foreskin through the dermis as described hereafter.

The nutrient medium is any composition which maintains the viability ofthe foreskin. Preferably, the nutrient medium has a liquid phase such asa solution, suspension or emulsion. A portion of the medium may beobtained commercially, such as Iscove's modified Dulbecco's medium(IMDM), Ham's nutrient mixture F-10 medium, Minimum essential media(MEM), RPMI media 1630 or 1640, Dulbecco's Modified Eagle Media (D-MEM)or Media 199 all of which are manufactured by Gibco Laboratories ofGrand Island, N.Y. as well as other companies, the specification sheetsof which are hereby incorporated by reference. Additionally the mediumcomprises about 10% to about 30% horse serum and about 2% to about 10%fetal bovine serum; the serums may be purchased from Hyclone Lab Inc.,of Logan, Utah. If necessary, an alkalizer such as sodium bicarbonatemay be added until the medium achieves a preferred pH, preferably abouta physiological pH. Antibiotics such as penicillin and/or streptomycin,may also be added for microbial control.

If transportation of the foreskin is necessary after surgical removal,the foreskin is immediately placed on an absorbent support saturatedwith the nutrient medium. In order to maintain the viability of theforeskin, the foreskin is disposed in the medium within about 3-4 hoursafter surgical removal. The position of the foreskin in the mediumshould be that the dermis contacts the medium and the epidermis is notsubstantially contacted by the medium.

Referring to FIGS. 1 and 2 of the organ culture system 10, the presentinvention comprises a foreskin 12, having an epidermis 14 and a dermis15. The foreskin 12 is placed in the medium 16 so that the medium 16contacts the dermis 15 but does not substantially contact the epidermis14. The epidermis 14 is in contact with the environment as under in vivoconditions. The medium 16 nourishes the foreskin 12, and maintains itsviability while the organ culture is under study. About 7 days or lessare generally needed to complete such a study.

The medium 16 comprises a liquid phase and is disposed in a containersuch as a petri dish 26. The amount of medium needed will depend upon,among other things, the container and the support means used. Generallyabout 5 ml to about 10 ml is a sufficient amount of medium for oneforeskin in a petri dish having a diameter of 1.5 inches with theforeskin supported on a float as described hereafter.

The foreskin 12 may be supported by any means which will permitpositioning of the foreskin in the medium 16 as previously described.Preferably, the foreskin 12 is disposed on a buoyant and permeable float18 having a top 20 and a bottom 22; the dermis 15 contacts the top 20 ofthe float 18. The float 18 floats on top of the medium 16. The float 18preferably comprises a sterile plastic screen such as a Swinnex 25 cmfilter support screen manufactured by Millipore. More preferably, apermeable pad 24 is interposed between the dermis 15 and the top 20 ofthe float 18. The pad 24 functions to hold the foreskin 12 on the float18. One example of a pad 24 used in accordance with the presentinvention is a AP20 025 00 filter manufactured by Millipore. Becauseboth the float 18 and pad 24 are permeable to the culture medium 16, themedium maintains contact with the dermis 15 while the foreskin ispositioned on the float 18, as required to maintain the foreskinsviability. The float 18 is placed on top of the medium 16 and may freelymove about the top of the medium 16.

Once the foreskin is disposed in the organ culture system as describedherein, the organ culture system is incubated during the length ofobservation of the foreskin. Preferably the medium is changed daily,since nutrients may be depleted over time, and the incubation causesdegradation of medium components.

In using the human organ culture system of the present invention, theforeskin is surgically removed, prepared and disposed in the organculture system as described herein. Before positioning the foreskin inthe system, the foreskin should be observed to determine the amountand/or the condition of the biological factor under study to obtain abaseline measurement.

After treatment of the foreskin with an agent, the biological factor isagain observed for a post-treatment measurement to be compared to thebaseline measurement. For example, if the amount of tyrosinase in theforeskin is under study, the amount of tyrosinase is determined as abaseline measurement prior to application of the agent to the foreskin.If the toxicity of an agent on skin is to be studied, the foreskin maybe observed for DNA synthesis, protein synthesis, ATP synthesis, or anycombination of these cell functions. If the permeability of an agent onskin is to be determined, the absence of the agent in the skin is thebaseline measurement. Some examples of biological factors which may bestudied are tyrosinase, melanin, DNA, RNA, proteins, carbohydrates, orany other factor that can be measured or observed for differencesbetween the treated and the untreated foreskins.

If the foreskin is to be treated with an agent under study, the agentshould be added to the medium 16, so that it comes into contact with theforeskin 12 through the medium 16. Alternatively, the agent can beplaced directly on the epidermis 14. The treatment time will depend onthe results sought, the identity of the agent under study, the time overwhich the foreskin can remain viable, and other variables.

After the foreskin has been treated with the agent under study, theforeskin may be observed and/or tested in any manner which willdetermine the differences in the foreskin from the observation and/ortests on the untreated foreskin. For example, the activity of tyrosinasemay be measured as described herein and correlated to an increase inmelanogenesis; the rate of DNA synthesis can be measured by ³H-thymidine uptake and compared to controls; or the increase or decreasein the synthesis of proteins and/or RNA can be measured by determiningthe rate of incorporation of [³ H] leucine (protein) or [³ H] uridine(RNA) into acid-precipitating material.

From the foregoing, it will be appreciated that the organ culture systemof the present invention offers an opportunity to study the effects ofmany agents on human skin, and many biochemical processes of human skin.Factors other than pigmentation processes, and agents other thanpigmentation enhancers may be evaluated for effects on the skin. Forexample, cosmetic agents or suspected toxic environmental compounds maybe tested for their effects on skin morphology, DNA synthesis, abnormalchromosome appearance, skin cell viability, and protein and RNAsynthesis. Also, the permeability of agents through the epidermis andthe dermis may be studied in order to evaluate agents for use intransdermal delivery systems for systemic delivery of the agent.

The following examples illustrate the practice of the method andcomposition of the present invention:

EXAMPLE 1 Preparation of Human Foreskin

At the time of surgical removal, human foreskins were placed on sterilegauze saturated with sterile IMDM medium (Iscove's modified Dulbecco'smedium purchased from Irvine Scientific of Santa Ana, Calif.) fortransportation from the Hospital nursery to the laboratory. The tissueswere rinsed in sterile IMDM medium containing 500 U/ml penicillin and500 μg/ml of streptomycin for 5 minutes. Under sterile conditions, anintradermal injection of medium was performed from the dermal side priorto dissection of the mucous membrane and lower dermis by scissors tomake the thickness of skins equal. The foreskins were then cut intoapproximately 3 mm×3 mm squares and either frozen at -75° C. or placedin organ culture as described hereafter.

EXAMPLE 2 Organ Culture

The organ culture medium was prepared from IMDM with glutaminesupplemented with 20% horse serum, 5% fetal bovine serum, 100 U/mlpenicillin, 100 μg/ml streptomycin and 3 mg/ml sodium bicarbonate. Theserums were obtained from Hyclone Lab. Inc. of Logan Utah. Culture unitswere prepared by placing sterilized filters (AP20 025 00, Millipore)over sterilized support screens (25 cm Swinnex filter support screens,Millipore) in the wells of 6-well tissue culture plates (Falcon 3046)with medium added to the wells such that the skin support screensfloated and the filter absorbed the medium from beneath. The tissuesamples were placed, epidermis up, on top of the saturated filters andincubated at 37° C. in a 5% CO₂ humidified atmosphere. The medium waschanged everyday. Harvested cultures were frozen at -75° C.

EXAMPLE 3 Light Microscopy

For histological study, thawed samples of fresh tissue and explants weremounted in OCT compound (ICN Immuno Biologicals, Lisle, Ill.) and frozenby liquid nitrogen. Cryostat sections (6 μm thick) were fixed in 2%formaldehyde for 2 hours at 40° C., and then stained either withhematoxylin and eosin or subjected to dopa staining. The dopa reactionswere carried out by incubation in two changes of 0.1% L-dopa solutionbuffered to pH 7.4 in 0.1M sodium phosphate buffer for 4 hours at 37° C.

EXAMPLE 4 Determination of Tyrosinase Activity

Tyrosinase activity in human skin organ cultures was determined bymeasuring the tyrosine hydroxylase activity of the enzyme. The assaymeasures the production of ³ H₂ O during the conversion of [³ H]tyrosineto L-DOPA. Weighed skin preparations were incubated in 0.3 ml of areaction mixture containing 0.01 mM of L-tyrosine, 5-6 uCi/ml of [³ H]tyrosine and 0.1 mM L-DOPA in 0.1M of pH 6.8 phosphate buffer for 4hours at 37° C. To terminate the reaction, 1 ml of phosphate buffer wasadded, the tubes vortexed, and 0.4 ml aliquots removed in triplicate andmixed with an equal volume of Norit SG activated charcoal (10% w/v, in0.1N HCl). Following centrifugation at 2000 xg for 10 min, thesupernatants (0.5 ml) were placed in scintillation vials, scintillationfluid added, and vials counted in a TM Analytic 6895 scintillationcounter equipped with a DPM processor.

EXAMPLE 5 Evaluation of Viability of the Human Foreskin

In order to assess the efficacy of the culture system, both black andwhite human foreskins were placed in culture and at 24 hours intervalsskin sections were removed for histological examination by H&E stainingand by dopa staining. The human foreskin retained its normal morphologythroughout the 7 day length of the study. There was no difference instructural viability between black and white skin. When the dopa oxidaseactivity of cultured skin melanocytes was examined by dopa staining, asimilar staining pattern between day 0 and day 7 foreskin cultures wasobserved, showing that the culture conditions maintain the viability ofthe skin for many days. Foreskins placed in culture within 3-4 hours ofsurgical removal showed normal structural integrity throughout theculture period whereas samples placed in culture more than 4 hours afterremoval did not maintain structural integrity.

When tyrosinase activity levels were determined in cultured skin, amarked decline in activity during the first 24 hours in culture in bothblack and white foreskins was observed. After this initial drop,however, tyrosinase activity stabilized for the remainder of the 7 dayincubation period.

EXAMPLE 6 Evaluation of Foreskin Treatment

Foreskins treated with α-MSH (melanocyte stimulating hormone from Sigma,St., Louis, Mo.) showed an increase in tyrosinase activity whereas.,foreskins treated with D-phe-MSH (Sigma, St. Louis, Mo.) showed a markedstimulation of tyrosinase. This shows that the organ culture system canrespond to hormones.

A stronger stimulation of tyrosinase was found with theophylline. Humanforeskins were treated with the compounds shown in Table 1, placed inculture, and incubated for 72 hours. The skins were then assayed fortyrosinase activity with the tyrosine hydroxylase assay method asdescribed herein. As shown in Table 1, dibutyryl cAMP was less effectivein stimulating tyrosinase activity.

Table 2 shows the activity of theophylline in a pharmaceutical topicalcarrier. Organ cultures were incubated with the compounds shown in Table2 for 72 hours. The medium and compounds were changed daily. Tyrosinaseactivity was determined by measuring the tyrosine hydroxylase activityof the enzyme.

Human foreskin organ cultures were treated with the compounds listed inTable 3 for 3 days and tyrosinase activity determined as describedherein. Theophylline was used at 1 mM.

                  TABLE 1                                                         ______________________________________                                        Summary of effects of hormones and other compounds                            on tyrosinase activity in human foreskin organ cultures.                                Tyrosinase Activity                                                 Treatment   Stimulated     Unaffected                                         ______________________________________                                        Theophylline                                                                              11B(92%); 6W(86%)                                                                            1B(8%) ;1W(14%)                                    (1 mM)                                                                        MSH (2 × 10.sup.-7 M)                                                                2B(33%); 2W(40%)                                                                            4B(67%);3W(60%)                                    dbcAMP (0.1 mM).sup.a                                                                      3B(33%); 1W(25%)                                                                            6B(67%);3W(75%)                                    D-phe-MSH    2B(50%); 2W(50%)                                                                            2B(50%);2W(50%)                                    (10.sup.-8 M)                                                                 ______________________________________                                         .sup.a dbcAMP is dibutyryl cAMP. B = black; W = white;.                       Values are the averages of triplicate determinations ± s.d.           

                  TABLE 2                                                         ______________________________________                                        Effect of IBMX (isobutyl methylxanthine) and theophylline in                  vehicle.sup.a on tyrosinase activity in human foreskin organ cultures.                      Tyrosinase Activity                                             Treatment     pmoles .sup.3 H.sub.2 O/hour/mg skin                            ______________________________________                                        control       3.15 ± 0.1                                                   IBMX (0.1 mM) 7.03 ± 0.49                                                  Theophylline/ 6.32 ± 0.22                                                  vehicle (1 mM)                                                                ______________________________________                                         .sup.a vehicle consists of 20% water, 50% isopropanol and 30% glycerol.       Values are the averages of triplicate assays ± s.d.                   

                  TABLE 3                                                         ______________________________________                                        Effect of Hormones and other compounds on tyrosinase activity in              human foreskin organ cultures.                                                Skin              Tyrosinase Activity                                                                            Percentage                                 Type   Treatment  pmoles .sup.3 H.sub.2 O/hr/mg skin                                                             of control                                 ______________________________________                                        Black  control    2.55 ± 0.13   212                                               theophylline                                                                             5.40 ± 0.26                                              Black  control    3.09 ± 0.12   197                                               theophylline                                                                              6.1 ± 0.24                                              White  control    0.31 ± 0.03   307                                               theophylline                                                                             0.95 ± 0.05                                                     D-phe-MSH  0.51 ± 0.05   165                                               (10.sup.-9 M)                                                          Hispanic                                                                             control    0.41 ± 0.02   349                                               D-phe-MSH  1.43 ± 0.02                                                     (10.sup.-8 M)                                                                 D-phe-MSH  0.91 ± 0.03   222                                               (10.sup.-9 M)                                                          ______________________________________                                         Values are the averages of triplicate determinations ± s.d.           

Changes may be made in the combination and arrangement of parts,elements, steps, compositions and procedures described herein withoutdeparting from the spirit and scope of the invention as defined in thefollowing claims.

What is claimed is:
 1. A method of promoting the tanning of subject'sskin, comprising:treating the skin with a composition of matter forstimulating melanogenesis in melanocytes of human epidermis, thecomposition disposed in a pharmaceutical carrier capable of deliveringthe composition to the melanocytes under in vivo conditions, thecomposition comprising:an effective amount of a methylxanthine effectivein increasing tyrosinase activity in the melanocytes to an amountsufficient to result in increasing levels of melanin in the melanocytesresulting in tanning of the epidermis; and an effective amount oftyrosine for serving as a tyrosinase substrate.
 2. A method forpromoting the tanning of a subject's skin, comprising:treating the skinwith a composition of matter for stimulating melanogenesis inmelanocytes of human epidermis, the composition disposed in apharmaceutical carrier capable of delivering the composition to themelanocytes under in vivo conditions, the composition comprising: aneffective amount of a methylxanthine effective in increasing tyrosinaseactivity in the melanocytes to an amount sufficient to result inincreasing levels of melanin in the melanocytes resulting in tanning ofthe epidermis.
 3. A method of promoting the tanning of a subject's skin,comprising;treating the skin with a composition of matter forstimulating melanogenesis in melanocytes of human epidermis, thecomposition disposed in a pharmaceutical carrier capable of deliveringthe composition to the melanocytes under in vivo conditions, thecomposition comprising:an effective amount of a phosphodiesteraseinhibitor effective in increasing tyrosinase activity in the melanocytesto an amount sufficient to result in increasing levels of melanin in themelanocytes resulting in tanning of the epidermis; and an effectiveamount of tyrosine for serving as a tyrosinase substrate.
 4. A methodfor promoting the tanning of a subject's skin, comprising:treating theskin with a composition of matter for stimulating melanogenesis inmelanocytes of human epidermis, the composition disposed in apharmaceutical carrier capable of delivering the composition to themelanocytes under in vivo conditions, the composition comprising:aneffective amount of a phosphodiesterase inhibitor effective inincreasing tyrosinase activity in the melanocytes to an amountsufficient to result in increasing levels of melanin in the melanocytesresulting in tanning of the epidermis.
 5. A method for promoting thetanning of a subject's skin, comprising:treating the skin with acomposition of matter for stimulating melanogenesis in melanocytes ofhuman epidermis, the composition disposed in a pharmaceutical carriercapable of delivering the composition to the melanocytes under in vivoconditions, the composition comprising:an effective amount oftheophylline effective in increasing tyrosinase activity in themelanocytes to an amount sufficient to result increasing levels ofmelanin in the melanocytes resulting in tanning of the epidermis.
 6. Themethod of claim 5 wherein the pharmaceutical carrier comprises analcohol.
 7. A method for promoting the tanning of a subject's skin,comprising:treating the skin with a composition of matter forstimulating melanogenesis in melanocytes of human epidermis, thecomposition disposed in a pharmaceutical carrier capable of deliveringthe composition to the melanocytes under in vivo conditions, thecomposition comprising:an effective amount of isobutylmethylxanthineeffective in increasing tyrosinase activity in the melanocytes to anamount sufficient to result in increasing levels of melanin in themelanocytes resulting in tanning of the epidermis.
 8. The method ofclaim 7 wherein the pharmaceutical carrier comprises an alcohol.
 9. Amethod of promoting the tanning of a subject's skin, comprising:treatingthe skin with a composition of matter for stimulating melanogenesis inmelanocytes of human epidermis, the composition disposed in apharmaceutical carrier capable of delivering the composition to themelanocytes under in vivo conditions, the composition comprising:anamount of theophylline and isobutylmethylxanthine effective inincreasing tyrosinase activity in the melanocytes to an amountsufficient to result in increasing levels of melanin in the melanocytesresulting in tanning of the epidermis.
 10. The method of claim 9 whereinthe pharmaceutical carrier comprises an alcohol.